ghent university - genetic differentiation of artemia ...all the artemia cyst samples were available...

ISSN 2320-5407 International Journal of Advanced Research (2014), Volume 2, Issue 4 ,1154-1164

1154

Journal homepage: http://www.journalijar.com INTERNATIONAL JOURNAL

OF ADVANCED RESEARCH

RESEARCH ARTICLE

Genetic differentiation of Artemia franciscana (Kellogg, 1906) in Kenyan coastal saltworks

Erick Ochieng Ogello

1*, Betty M. Nyonje

2 and Gilbert Van Stappen

3

1. 1*Kenya Marine and Fisheries Research Institute, Kegati Aquaculture Research Station P.O. Box 3259 – 40200,

Kisii, Kenya

2. 2Kenya Marine and Fisheries Research Institute, Mombasa Research Centre P. O. Box 81651 Mombasa, Kenya

3. 3Laboratory of Aquaculture & Artemia Reference Center (ARC), Ghent University, Rozier 44, B-9000 Gent,

Belgium

.

Manuscript Info Abstract

Manuscript History:

Received: 12 February 2014 Final Accepted: 22 March 2014

Published Online: April 2014

Key words: mtDNA, RFLP, Heat shock protein,

Kenya, Artemia franciscana

*Corresponding Author

Erick Ochieng Ogello

The nature of genetic divergence between the Artemia population native to

San Francisco Bay, (SFB) USA and those from the introductions of SFB

material in the Kenyan coast two decades ago were investigated using the

mitochondrial DNA (mtDNA) and heat shock protein 70 (Hsp70) gene

molecular markers. The DNA was extracted from 80 single Artemia cysts

using the Chelex protocol. The 1,500 bp fragment of the 12S - 16S region of

the mtDNA and a 1,935 bp fragment of the Hsp70 gene were amplified

through Polymerase Chain Reaction (PCR) followed by Restriction

Fragment Length Polymorphism (RFLP) digestion using appropriate

endonucleases. The mtDNA analysis indicated higher haplotype diversity

(0.76 ± 0.07) in Artemia from Fundisha saltworks while the rest of the

samples were monomorphic. A private haplotype (AAABBA) in Fundisha

samples confirmed a molecular evidence of a systematic genetic

differentiation albeit in an insignificant manner (P > 0.05). There was

molecular evidence of coexistence of SFB and GSL Artemia strains in

Fundisha saltworks. The monomorphic DNA fingerprint in Kensalt Artemia

cysts was probably caused by non-sequential Artemia culture system and

limited mtDNA fragment size analysed. The Hsp70 gene RFLP fingerprint

did not show any unique gene signatures in the Kenyan Artemia samples

suggesting that other factors other than Hsp70 were involved in their superior

thermotolerance. Further genetical studies based on the larger mtDNA

fragment using robust genetic markers are recommended. Ecological studies

of the heat shock protein family and the stress response would be more

relevant than the qualitative RFLP technique.

Copy Right, IJAR, 2014,. All rights reserved

Introduction The brine shrimps Artemia are small crustaceans adapted to live in stressful environmental conditions of hypersaline

habitats such as salt lakes, coastal lagoons and solar saltworks, where they feed primarily on phytoplankton and

bacteria (Persone and Sorgeloos, 1980; Toi et al., 2013). Being osmotolerant animals, Artemia can withstand

habitats whose salinity levels range between 10 - 340 g L-1

with fluctuating ionic composition and temperature

profiles (Van Stappen, 2002). Artemia adaptation to these conditions has occurred at molecular, cellular,

physiological and population level making Artemia fit to survive and reproduce effectively in such insulting

environments (Gajardo and Beardmore, 2012). Artemia has high genetic variability (Kappas et al., 2004) that makes

them model animals for studying evolutionary processes such as genetic differentiation, which indeed, is the focus

of this paper. The most discussed reproductive adaptation mechanism of the genus Artemia is the existence of two

distinctly short cycles of development (Clegg et al., 2004; Kappas et al., 2004). During favourable environmental

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conditions, an ovoviviparous reproduction cycle occurs where the adult females produce the free swimming naupli

(Anderson et al., 1970). However, during stressing environmental conditions, an oviparous reproductive cycle

prevails and the adult female Artemia produces metabolically inactive cysts as the parental animals dies (Dutrieu,

1960; Van Stappen, 1996). When conducive environmental conditions return, the cysts hatch into free swimming

nauplii in a process that lasts for about 20 hours, thus completing the cycle (Pearson and Sorgeloos, 1980; Van

Stappen, 1996).

The ability of the brine shrimp Artemia to inhabit hypersaline environments gives them a wide global geographical

representation (Persoone and Sorgeloos, 1980). In fact, as Triantaphyllidis et al. (1998) put it; the only place where

Artemia cannot be found is Antarctica. So far, discrete Artemia populations have been identified in about 600

natural salt lakes and saltworks and further survey efforts are still on course to identify more Artemia biotopes all

over the world (Van Stappen, 2002).

For a long time, Artemia morphometric features have been used to discriminate between different populations

despite many human errors (Naceur et al., 2010). Today, Artemia phylogeny can be easily verified and cyst samples

scientifically authenticated thanks to molecular techniques (Bossier et al., 2004; Van Stappen, 2008). According to

Avise (2004), molecular techniques provide full access to unlimited pool of organism’s genetic variability. The

extensive study of inter- and intra-specific diversity of Artemia has been made possible due to a variety of nuclear

and mitochondrial DNA markers for instance ITS-1, Hsp26, COI, 12S and 16S mtDNA (Perez et al., 1994; Hou et

al., 2006) and tools such as Restriction Fragment Length Polymorphism (RFLP) (Bossier et al., 2004; Gajardo et al.,

2004; Eimanifar et al., 2006), Random Amplified Polymorphic DNA (RAPD) (Sun et al.,1999a; Camargo et al.,

2002), Amplified Fragment Length Polymorphism (AFLP) (Sun et al., 1999b) using either single or pooled

individuals or cysts samples (Kappas et al., 2004). Other tools include microsatellites and Single Strand

Conformation Polymorphism (SSCP) (Blouin et al., 1996).

In the RFLP molecular technique, the targeted DNA genome size is first PCR amplified before digestion with

restriction enzymes. The digested product is then separated according to their size by agaorose gel electrophoresis

(Eimanifar et al., 2006). The mitochondrial genome of A. franciscana is estimated to be 15,822 nucleotides long

(Valverde et al., 1994). The mtDNA is highly conserved compared to nuclear DNA, making it a robust marker for

tracking animals’ ancestry (Krieg et al., 2000). Eimanifar et al. (2006) found genetic nucleotide divergence within

Artemia populations found in different ecological zones of Lake Urmia using the RFLP method. Agh et al. (2009)

showed that bisexual A. urmiana and parthenogenetic populations in Iran are genetically close based on RFLP of

their 1,500 bp mtDNA fragment. Manaffar (2012) also conducted an RFLP analysis of the 1,500 bp mtDNA

fragment on A. urmiana cysts and detected high polymorphism among cysts from different stations in Urmia Lake.

Through RFLP analysis of a 1,500 bp mitochondrial rDNA fragment, Bossier et al. (2004) developed a methodology

to authenticate Artemia cyst samples. Kappas et al. (2004) investigated how A. franciscana native to SFB colonised

unfamiliar Vietnam environments through RFLP technique based on the 2,963 bp long mtDNA target sequence.

Unique genetic signatures were observed in the mtDNA genome of the Vietnam Artemia strain suggesting a process

of strong selective pressure in them (Kappas et al., 2004).

There is much information regarding Artemia’s ability to synthesize heat shock proteins, such as Hsp26 and Hsp70

(Clegg et al., 2001; Crack et al., 2002; Willsie and Clegg, 2002). Artemia cysts contain substantial amounts of heat

shock proteins because they are the surviving agents in stressful environments (Clegg et al., 1999; Van Stappen,

2002). Scientific evidence has proven that the family of heat shock proteins are critical for thermal resistance

(Frankenberg et al., 2000), desiccation tolerance (Ma et al., 2005) and reduces osmotic stress (DuBeau et al., 1998;

Todgham et al., 2005). Therefore Hsp70 protects organisms against multidimensional environmental challenges.

Clegg et al. (2001) found that Artemia cysts produced in hotter environments contain higher amounts of heat shock

proteins such as artemin, p26 and Hsp70. Therefore, the stress proteins could be involved in the adaptation of A.

franciscana from SFB growing in the much hotter environments such as salt ponds in Vietnam (Clegg et al., 2001)

and probably Kenyan coastal areas.

Between 1984 and 1986, a non-native A. franciscana was introduced along the Kenyan coast (Fundisha and Kurawa

salt farms). Today, the A. franciscana has permanently colonised the Kenyan coast, where about eight saltworks

exist today. Since 2009, Fundisha saltworks has been re-inoculated using GSL Artemia strains suggesting

coexistence of GSL and SFB Artemia strains. This is a subject that can only be revealed through molecular studies.

The laboratory culture experiments of Mremi (2011) and Kapinga (2012) showed that Kenyan Artemia are superior

to their original SFB inoculants in terms of reproductivity and thermotolerance at elevated temperatures. However,

no information is available on their genetic architecture to support these phenotypic characteristics. To date, it is not

known whether SFB and GSL Artemia strains coexist in Fundisha saltworks. Neither do we know the genetic micro-

evolutionary divergences that have occurred in the Kenyan Artemia population. Artemia population have also been

discovered in Tanga region (Tanzania). However, no scientific information is available about them. The


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polymorphic analysis of the 1,935 bpHsp70 gene was considered to add more perspective on the much anticipated

genetic adaptation levels of the Kenyan Artemia populations. The hypothesis of this study was that the genetic

pattern of the Kenyan Artemia strains would be mutually polymorphic. The present laboratory based study aimed to

genetically characterize the Kenyan Artemia cysts based on the mitochondrial DNA and heat shock protein 70

(Hsp70) genes. The study also determined the purity of Artemia populations in Kensalt and Fundisha saltworks and

established the genetic relationships between the Kenyan and Tanga (Tanzania) Artemia cysts.

Materials and methods Source of Artemia cyst samples and study area

A total of 80 individual Artemia cysts, 10 replicates from each of 8 samples were used in the study. The 8 samples

included 4 samples (Fundisha, Ken1, Ken2 and Ken3) harvested between 1996 and 2012 from selected salt farms at

the Kenyan coast, located at located at 3° 50' 0" South, 39° 46' 0" East (Study area Fig. 1). One sample from Great

Salt Lake, Utah state in USA (GSL), another sample from San Francisco Bay (SFB) USA and one sample from Vinh

Chau (VC), Vietnam, were used as controls. Artemia samples from Tanga (Tanzania) were also analysed as

additional study. All the Artemia cyst samples were available at the Laboratory of Aquaculture & Artemia

Reference Center (ARC), Ghent University, Belgium where they were stored at 4oC in the cyst bank. The selection

criteria for the Kenyan Artemia cyst followed Nyonje (2011) report. Cysts from Kensalt farm (Ken1, Ken2, Ken3),

which were different batches of the same population, were considered because of their known reproductive

characteristics (Mremi, 2011; Kapinga 2012) while the much hypothesised coexistence of SFB and GSL Artemia

strains in Fundisha saltwork was the reason for considering cysts from there. The use of cysts for DNA extraction

for mtDNA and Hsp70 analysis were preferred to individual Artemia adults to prevent loss of genetic information

due to selective hatching (Van stappen 2008).

DNA extraction

DNA was extracted from single Artemia cysts using the Chelex method (Walsh et al., 1991). The cyst was isolated

using a sterile 10 μL pipette point and transferred to a sterile eppendorf (1.7 mL) where 30 μL of milliQ water (PCR

water) was added and left to hydrate for 1 hr. In each eppendorf tube, the cyst was crushed using a sterile pellet

pestle (Sigma-Aldrich Z35997-1EA) before adding 30 μL of well homogenized 10% Chelex slurry (Chelex-100 -

Biorad, Belgium). The samples were vortexed for 10 - 15 s before spinning for 1 min at 13,000 rpm in a micro-

centrifuge. The samples were incubated for 20 min at 95oC, vortexed again for 10 - 15 s and further spanned at

13,000 rpm for 1 min. The quantity of the extracted DNA was measured using a NanoDrop® ND-1000 machine

while the quality of the DNA was verified through agarose gel electrophoresis (Lind et al., 2006).

PCR amplification of the 1,500 bp 12S - 16S mtDNA fragment

The double stranded DNA amplification was performed in 50 µL reaction volumes each containing a mixture of

36.375 µL PCR water, 5 µL 10 x Taq buffer + KCl-MgCl2, 5 µL MgCl2 (25 mM solution), 1 µL dNTP (10 mM

each), 1 µL of primer 1 and 2 (work-solution), 0.125 µL BSA, 0.5 µL Taq-polymerase and 2 µL of approximately 5

- 30 ng of template DNA extract, except for the negative control tube. The DNA samples were PCR amplified for

the 1,500 bp mtDNA fragment between the 12S - 16S region (Valverde et al., 1994). A combination of the forward

primer 12S - SP (5’-cta-gga-tta-gat-acc-cta-3’), and the reverse primer 16S - SP (5’- ccg-gtc-tga-act-cag-atc-3’) was

used according to Bossier et al. (2004). The BioRad PCR equipment was programmed such that the first cycle of

PCR reaction heated the mixture to 94oC for 2 minutes to activate the Taq polymerase enzyme. This was followed

by 34 cycles of: 1) a denaturing step at 94oC for 1 min; 2) an annealing phase at 52

oC for 45 seconds; 3) an

elongation phase at 72oC for 2 min and the final elongation cycle step at 72

oC for 4 minutes.

PCR amplification of the Hsp70 gene

The PCR reaction mixture of 25 µL contained 16.32 µL PCR water, 4.8 µL 10 x GoTaq buffer + KCl-MgCl2, 1.2 µL

MgCl2 (25 mM solution), 0.48 µL dNTP (10 mM each), 0.48 µL of primer 1 and 2 (work-solution), 0.24 µL GoTaq-

polymerase and 1 µL of approximately 150 ng of template DNA extract except for the negative control tube. The

DNA samples were assayed for PCR amplification of the 1,935 bp Hsp 70 gene fragment (Baruah et al., 2010). A

combination of the forward primer Hsp70forward (5’-cac-cat-ggc-aaa-ggc-acc-agc-aat-agg-3’) and the reverse primer

Hsp70reverse (5’-ata-gtt-ggg-cca-ctg-cct-gtt-cca-g-3’) were used (Baruah et al., 2010). The PCR conditions were

modified from Baruah et al. (2010) as follows: denaturation step at 94oC for 5 min followed by 35 cycles of 95

oC for

1 min, annealing at 63oC for 1 min and elongation at 72

oC for 4 min followed by a final extension step for 10 min at

72oC.


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Restriction digestion: RFLP procedure

The amplified 1,500 bp mtDNA fragments were screened for polymorphism using six restriction endonucleases

(AluI, HaeIII, HinfI, RsaI, XbaI and HpaII) (Bossier et al., 2004; Kappas et al., 2004). The reactions were done

according the manufacturer’s instructions (see Tab. 1). For each reaction tube, a total reaction volume of 23.5 µL

consisted of 16 µL PCR water, 2 µL Tango buffer, 0.5 µL of enzyme and 5 µL of PCR amplified DNA product.

Digested products were electrophoretically separated on 2 % agarose gel in a 1 X TAE buffer solution and stained

with 1 µL of GelRed. A voltage of 100 V was used to push the digested DNA fragments through the solidified

agarose gel for 1 h. A 100 bp promega DNA ladder was loaded as reference. A UV transilluminator was used to

visualise the fragments and photographed with a digital camera (Canon power shot G10).

For the Hsp 70 gene, restriction enzymes were selected based on the number of cleavage sites in the 1,935 bp

fragment of the Artemia franciscana nucleotide sequence (cDNA). Four restriction enzymes (Sau3A, Rsal, AluI and

HinfI) with recognition sequences GATC, GTAC, AGCT and GAATC respectively were used. In each reaction

tube, a total reaction volume of 23.5 µL contained 16 µL PCR water, 2 µL Tango buffer, 0.5 µL of enzyme and 5

µL of PCR amplified DNA product. The incubation temperature was 37oC while inactivation temperature was 65

oC

for 20 min for Sau3A enzyme. Gel-electrophoresis was as explained above but 1kb promega DNA ladder was

loaded as reference. The homologies of fragment patterns were established through side by side visual comparisons

for both mtDNA and Hsp70 gene.

Data analysis

The RFLP restriction pattern fragments were manually scored. Fragments less than 100 bp were neglected because

of technical inconsistencies. Unique endonuclease restriction patterns were identified by using specific letters. Each

cyst replicate was assigned a multi-letter code that described its composite mtDNA genotype haplotype. For each

sample, the haplotype frequency (hf) was manually calculated by counting the identical haplotypes and dividing by

the total replicates per sample (Nei, 1978). The mean haplotype frequency was calculated by adding all the

haplotype frequency in each haplotype then dividing by the total number of samples (Nei, 1987). The haplotype

diversity within samples was calculated based on Nei and Tajima’s (1981) formula.

Where: H = haplotype diversity; N = Sample size; x = haplotype frequency

The non-parametric Wilcoxon signed rank one sample t-test of SPLUS (Sportifire 2 + 8.2) statistical programme

was used to test significant difference among the sample’s haplotype frequencies at P = 0.05 level of significance.

The cluster dendrogram for samples was drawn using PyElph 1.4 software (Pavel and Vasile, 2012) based on the

unweighted average pair group method (UPGMA). For the RFLP of the Hsp70 gene, the sizes of the fragments were

only estimated by comparison with a 1kb ladder. No further data processing was done whatsoever for the Hsp70

RFLP marker.

Results

Based on A 260 / 280 index, some samples showed high quality DNA (Tanga, SFB, GSL and VC) while others had low

quality (Fundisha, Ken1, Ken2 and Ken3) (Table 2). Values of A 260 / 280 indexes between 1.7 and 2.0 indicate the

presence of pure DNA (Glasel, 1995).

H = N

N - 1

1 - ∑ x2

∑∑


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Fig. 1: Study area - map of the Kenyan coast showing the location of the salt belt and a more detailed

impression of the salt belt showing the individual salt producing companies in a North – South Orientation.

Fundisha saltwork is also called Crystalline

Table 1: The list and recognition sequences of the restriction enzymes used in the study including incubation

and activation temperatures as described by the manufacturer; N = C, G, T or A.

Enzyme Recognition sequence Incubation temperature Inactivation temperature

AluI 5’...A G C T...3’

3’...T C G A...5’

37O

C 65O

C / 20minutes

HaeIII 5’...G G C C...3’

3’...C C G G...5’

37O

C 80O

C / 20minutes

HinfI 5’...G A N T C...3’

3’...C T N A G...5’

37O

C 65O

C / 20minutes

RsalI 5’...G T A C...3’

3’...C A T G...5’

37O

C 80O

C / 20minutes

Xbal

HpaII

5’...T C T A G A...3’

3’...A G A T C T...5’

5’...C C G G...3’

3’...G G C C...5’

37O

C

37O

C

65O

C / 20minutes

80O

C / 20minutes

Indian Ocean

Fundisha/crystalline

Saltwork: 1,020 ha

Kensalt: 3,000 ha


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Table 2: Average quantity of DNA extracted from individual cysts from each sample including Artemia

Reference Centre (ARC) code. The DNA quantity was measured using a NanoDrop® ND-1000 machine.

Values are mean ± SE.

Sample ARC code DNA (ng /µL) A 260 / 280

Fundisha 1780 9.55 ± 0.69 2.12 ± 0.03

Ken1 1762 15.01 ± 0.43 1.59 ± 0.12

Ken2 1439 7.51 ± 0.45 3.04 ± 0.32

Ken3 1779 7.53 ± 0.63 2.99 ± 0.22

Tanga 1773 14.38 ± 0.70 1.99 ± 0.10

GSL 1768 27.99 ± 1.95 1.87 ± 0.02

SFB 1574 37.25 ± 1.26 1.92 ± 0.01

VC 1771 28.80 ± 1.75 2.01 ± 0.03

PCR amplification of the 1,500 bp 12S -16S mtDNA fragment

The primer combinations produced identical 1,500 bp fragments in all the 10 replicates in every sample analysed.

Only one replicate per sample is shown (Fig. 2)

Fig. 2: Example of agarose gel for PCR-amplified 1500 bp 12S – 16S mtDNA fragment for a single cyst per

sample. L: 1500 bp ladder; NC: Negative control.

RFLP analysis of the mtDNA The enzymes HaeIII and HpaII detected polymorphism only in the Fundisha sample (Fig. 3). The enzymes AluI,

XbaI, HinfI and RsaI were monomorphic across all samples (results not shown). In total, approximately 1,216

fragments were surveyed in the 1,500 bp 12S - 16S mtDNA target sequence. A total of three composite haplotypes

were identified in the mtDNA target sequence. All the three haplotypes were present in Fundisha Artemia samples

while the rest of the samples were monomorphic (Table 3). The most common haplotype was AAAAAA, being

detected in all the sample populations except GSL. This haplotype attained the highest frequency (0.4286) within

Fundisha samples. A private haplotype (AAABBA) was discovered in Fundisha sample while the haplotype

(AAAABA) was only shared between Fundisha and GSL (Table 3). The highest haplotype diversity (h) was

recorded in Fundisha cyst samples (0.76 ± 0.07).


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Figure 3: Example of agarose restriction fragment profile for the polymorphic HaeIII and HpaII enzymes on

Fundisha individual cyst samples. PCR-amplified 1500 bp of 12S – 16S mtDNA fragment for 7 single cyst

replicates per sample.

Table 3: Haplotype genotype frequencies, mean haplotype frequency (mhf), sample size, number of

haplotypes (nh) and haplotype diversity (h) in samples. Haplotype genotypes are denoted with capital letters,

each one corresponding to the restriction pattern obtained by a restriction enzyme in the following order;

AluI, Xbal, HinfI, HpaII, HaeIII and RsaI

The dendogram showed two major groups (GSL and SFB) while the Fundisha cyst samples appeared to be

intermediate (Fig.4).

Haplotype Haplotype

genotype

Samples

Fundisha Ken1 Ken2 Ken3 Tanga GSL SFB VC mhf

H1 AAAAAA 0.4286 1.0000 1.0000 1.0000 1.0000 0 1.0000 1.0000 0.80

H2 AAAABA 0.2857 0 0 0 0 1.0000 0 0 0.16

H3 AAABBA 0.2857 0 0 0 0 0 0 0 0.04

S. size 7 7 7 7 7 7 7 7

nh 3 1 1 1 1 1 1 1

h ± SD 0.76 ± 0.07 0.00 0.00 0.00 0.00 0.00 0.00 0.00

HaeIII

HpaII


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Figur 4: UPGMA dendrogram of Nei's genetic distance for 8 Artemia franciscana population samples. The

values on the horizontal lines stand for Neis genetic distances in percentage.

Molecular analysis of the Hsp70 gene fragment

The 1,935 bp Hsp70 gene fragment produced a non-polymorphic pattern in all the enzymes. Only the RFLP pattern

of enzyme Rsal is shown (Fig. 5). Interestingly, even restriction enzymes such as AluI and Sau3A with 7 and 6

cleavage sites respectively on the 1,935 bp Hsp70 gene fragment did not show any polymorphism in any of the

samples.

Figure 5: Agarose restriction fragment profile for the enzymes: Sau3A, AluI, HinfI and Rsal. The PCR

fragment was generated using DNA extracted from pooled Artemia cysts. L: 1Kb ladder, Lanes 1: Fundisha,

2: Ken1, 3: Ken2, 4: Ken3, 5: Tanga, 6: GSL, 7: SFB, 8: VC, 9: Undigested PCR product (2, 000 bp) control.

Discussion

The present study analysed mitochondrial DNA using the RFLP tool to detect polymorphism in the Kenyan A.

franciscana using DNA extracted from individual cysts. The monomorhic DNA fingerprints corresponded with zero


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genetic distance as shown by the UPGMA dendrogram (Fig. 4), indicating lack of genetic differentiation between

and among the Artemia samples. Lack of genetic diversity is risky in times of genetic bottleneck as the entire

population may perish. Ecological processes such as migration can cause high Artemia population heterogeneity in

the habitat but limited effective gene flow is observed (Hajirostamloo, 2009) because effective gene flow is much

slower compared to the process of dispersal (Naihong et al., 2000). The process of assortative mating can prevent

intercrossing even among coexisting Artemia species to reduce chances of speciation (Beristain et al., 2010). The

absence of genetic polymorphism within samples could have been due to the limited 1,500 bp fragment of mtDNA

analysed. Kappas et al. (2004) used a larger mtDNA fragment of 2,973 bp and detected significant genetic

polymorphism within the A. franciscana introduced in Vietnam almost 2 decades ago. Since Kenya and Vietnam

share similar Artemia inoculation history, one would have expected similar genetic evolutions. However, a larger

fragment has high chances of showing detailed microevolutionary changes that might not be detected in a limited

DNA fragment.

The environmental conditions are critical factors that may influence the Artemia population patterns and genetic

expressions (Evjemo and Olsen, 1999; Van Stappen, 2002). In Kenya, integrated salt - Artemia culture is a

continuous process where Artemia flourishes year round. In Vietnam, the saltworks are predictably sequential and

this favoured a faster evolution of VC Artemia strain (Kappas et al., 2004). This conforms to Manaffar’s (2012)

observation that genetic drift in the presence of limited gene flow facilitates the speciation process. Therefore, the

absence of periodical genetic bottlenecks in the Kenyan situation suggests that only natural selection process is

responsible for gene loss. Natural selection requires long time to cause meaningful genetic divergence (Gajardo and

Beardmore, 2012). Permanence and seasonality of the environment are key instruments driving considerable genetic

differentiation of Artemia leading to specific biota with definite genetic structures (Lenz, 1987). However, the

exclusive ovoviviparity of the New Zealand A. franciscana population (inoculated in 1950s) was due to genetic

differentiation caused by constant year-round salinity and temperature conditions (Wear and Haslett, 1986). The

mutations caused by high UV radiation have also been linked to genetic evolutionary changes in Artemia

populations (Hebert et al., 2002).

The current study might not have sufficiently assessed the samples intra-population diversity due to limited the

RFLP technique, which is inferior to detect intra-specific polymorphism (Bossier et al., 2004; Avise, 2004). The fact

that the single haplotype identified in Tanga samples was similar to Kensalt and Fundisha samples suggested they

are genetically close. This provides evidence that Artemia in tanga region was introduced by Kensalt management

who own saltworks there.

Based on the RFLP fingerprint pattern and the number of haplotype genotypes obtained in this study, only the

Artemia population in Fundisha saltworks was polymorphic albeit in an insignificant manner (P > 0.05). Therefore,

if indeed there was significant genetic differentiation between Kenyan (Kensalt) Artemia and their SFB ancestors,

then the tool used was not sufficiently adequate to detect this micro-evolutionary divergence. Nevertheless, the

private haplotype (AAABBA) in Fundisha cyst samples suggested a systematic genetic differentiation thus

molecular evidence of an existing subpopulation and genetic divergence from their SFB ancestors. The population-

specific haplotype identified in Fundisha saltwork may become useful in monitoring the geographic expansion of the

Artemia populations along the Kenyan coast. However, further studies using superior genetic tools like AFLP and

microsatellites are needed to authenticate this finding. There was molecular evidence of co-existence of both SFB

and GSL Artemia strains in Fundisha saltwork, conforming to Nyonje (2011) report. This finding is consistent with

the studies of Van Stappen (2002), who documented that coexistence of different Artemia strains or species within

the same site is a common scientific possibility.

The lack of genetic variation in the Hsp70 RFLP fingerprint pattern suggested that the samples analysed had the

same Hsp70 gene structure. Feder and Hofmann (1999) reported that little variation in the Hsp70 gene could be due

to the fact that it is evolutionary and functionally conserved. Based on Kapinga (2012) and Mremi (2011) findings, it

was hypothesised that the Kenyan Artemia posses unique Hsp70 gene signatures. Having rejected this hypothesis, it

means that factors other than the Hsp70 gene are responsible for the observed adaptations (thermotolerance). Future

studies should focus on more quantitative Hsp70 analysis such as western blot by chemiluminescence techniques

(Schutz-Geschwender et al., 2004).

Conclusions and recommendations

The mtDNA sequence analysis has provided some diagnostic power in comparing SFB, GSL and Kenyan Artemia

strains. Even though the genetic differentiation of the Kenyan Fundisha Artemia population from its SFB ancestors

is not statistically significant, the presence of a private haplotype genotype in Fundisha saltwork could be the

beginning of a long term micro-evolutionary process, which could lead to eventual geographic differentiation and


1163

progressive speciation of A. franciscana in the Kenyan environment. It may also help to explore and monitor future

expansion of the Artemia population. The Kenyan Kensalt Artemia population is not contaminated by other Artemia

strains while there is co-existence of SFB and GSL Artemia strains in Fundisha saltworks. Other factors other than

the Hsp70 family could be involved in the much cited thermotolerance superiority of the Kenyan Artemia

populations. More robust molecular markers targeting larger mtDNA fragment should be considered concurrently

with Hsp70 quantitative technique.

Acknowledgement

This study was funded by the Flemish Interuniversity Council, the Vlaamse Interuniversitaire Raad and the

University Development Cooperation (VLIR-UOS) through a joint project bringing together Kenya Marine &

Fisheries Research Institute (KMFRI) and Gent University, Laboratory of Aquaculture & Artemia Reference Center

(ARC).

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ghent university - genetic differentiation of artemia ...all the artemia cyst samples were available...

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